Bacteriophage (phage) display is a technique by which variant polypeptides are displayed as fusion proteins to the coat protein on the surface of bacteriophage particles (Scott, J. K. and Smith, G. P. (1990) Science 249: 386). The utility of phage display lies in the fact that large libraries of selectively randomized protein variants (or randomly cloned cDNAs) can be rapidly and efficiently sorted for those sequences that bind to a target molecule with high affinity. Display of peptide (Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378) or protein (Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363) libraries on phage have been used for screening millions of polypeptides for ones with specific binding properties (Smith, G. P. (1991) Current Opin. Biotechnol., 2:668). Sorting phage libraries of random mutants requires a strategy for constructing and propagating a large number of variants, a procedure for affinity purification using the target receptor, and a means of evaluating the results of binding enrichments. U.S. Pat. Nos. 5,223,409; 5,403,484; 5,571,689; 5,663,143.
Typically, variant polypeptides are fused to a gene III protein, which is displayed at one end of the viron. Alternatively, the variant polypeptides may be fused to the gene VIII protein, which is the major coat protein of the viron. Such polyvalent display libraries are constructed by replacing the phage gene III with a cDNA encoding the foreign sequence fused to the amino terminus of the gene III protein. This can complicate efforts to sort high affinity variants from libraries because of the avidity effect; phage can bind to the target through multiple point attachment. Moreover, because the gene III protein is required for attachment and propagation of phage in the host cell, e.g., E. coli, the fusion protein can dramatically reduce infectivity of the progeny phage particles.
To overcome these difficulties, monovalent phage display was developed in which a protein or peptide sequence is fused to a portion of a gene III protein and expressed at low levels in the presence of wild-type gene III protein so that particles display mostly wild-type gene III protein and one copy or none of the fusion protein (Bass, S. et al. (1990) Proteins, 8:309; Lowman, H. B. and Wells, J. A. (1991) Methods: a Companion to Methods in Enzymology, 3:205). Monovalent display has advantages over polyvalent phage display in that progeny phagemid particles retain full infectivity. Avidity effects are reduced so that sorting is on the basis of intrinsic ligand affinity, and phagemid vectors, which simplify DNA manipulations, are used. See also U.S. Pat. No. 5,750,373 and U.S. Pat. No. 5,780,279. Others have also used phagemids to display proteins, particularly antibodies. U.S. Pat. Nos. 5,667,988; 5,759,817; 5,770,356; and 5,658,727.
A two-step approach has been used to select high affinity ligands from peptide libraries displayed on M13 phage. Low affinity leads were first selected from naive, polyvalent libraries displayed on the major coat protein (protein VIII). The low affinity selectants were subsequently transferred to the gene III minor coat protein and matured to high affinity in a monovalent format. Unfortunately, extension of this methodology from peptides to proteins has been difficult. Display levels on protein VIII vary with fusion length and sequence. Increasing fusion size generally decreases display. Thus, while monovalent phage display has been used to affinity mature many different proteins, polyvalent display on protein VIII has not been applicable to most protein scaffolds.
Although most phage display methods have used filamentous phage, lambdoid phage display systems (WO 95/34683; U.S. Pat. No. 5,627,024), T4 phage display systems (Ren, Z-J. et al. (1998) Gene 215:439; Zhu, Z. (1997) CAN 33:534; Jiang, J. et al. (1997) can 128:44380; Ren, Z-J. et al. (1997) CAN 127:215644; Ren, Z-J. (1996) Protein Sci. 5:1833; Efunov, V. P. et al. (1995) Virus Genes 10:173) and T7 phage display systems (Smith, G. P. and Scott, J. K. (1993) Methods in Enzymology, 217, 228-257; U.S. Pat. No. 5,766,905) are also known.
Many other improvements and variations of the basic phage display concept have now been developed. These improvements enhance the ability of display systems to screen peptide libraries for binding to selected target molecules and to display functional proteins with the potential of screening these proteins for desired properties. Combinatorial reaction devices for phage display reactions have been developed (WO 98/14277) and phage display libraries have been used to analyze and control bimolecular interactions (WO 98/20169; WO 98/20159) and properties of constrained helical peptides (WO 98/20036). WO 97/35196 describes a method of isolating an affinity ligand in which a phage display library is contacted with one solution in which the ligand will bind to a target molecule and a second solution in which the affinity ligand will not bind to the target molecule, to selectively isolate binding ligands. WO 97/46251 describes a method of biopanning a random phage display library with an affinity purified antibody and then isolating binding phage, followed by a micropanning process using microplate wells to isolate high affinity binding phage. The use of Staphylococcus aureus protein A as an affinity tag has also been reported (Li et al. (1998) Mol. Biotech., 9:187). WO 97/47314 describes the use of substrate subtraction libraries to distinguish enzyme specificities using a combinatorial library which may be a phage display library. A method for selecting enzymes suitable for use in detergents using phage display is described in WO 97/09446. Additional methods of selecting specific binding proteins are described in U.S. Pat. Nos. 5,498,538; 5,432,018; and WO 98/15833.
Methods of generating peptide libraries and screening these libraries are also disclosed in U.S. Pat. Nos. 5,723,286; 5,432,018; 5,580,717; 5,427,908; and 5,498,530. See also U.S. Pat. Nos. 5,770,434; 5,734,018; 5,698,426; 5,763,192; and 5,723,323.
Methods which alter the infectivity of phage are also known. WO 95/34648 and U.S. Pat. No. 5,516,637 describe a method of displaying a target protein as a fusion protein with a pilin protein of a host cell, where the pilin protein is preferably a receptor for a display phage. U.S. Pat. No. 5,712,089 describes infecting a bacteria with a phagemid expressing a ligand and then superinfecting the bacteria with helper phage containing wild type protein III but not a gene encoding protein III followed by addition of a protein III-second ligand where the second ligand binds to the first ligand displayed on the phage produced. See also WO 96/22393. A selectively infective phage system using non-infectious phage and an infectivity mediating complex is also known (U.S. Pat. No. 5,514,548).
Phage systems displaying a ligand have also been used to detect the presence of a polypeptide binding to the ligand in a sample (WO/9744491), and in an animal (U.S. Pat. No. 5,622,699). Methods of gene therapy (WO 98/05344) and drug delivery (WO 97/12048) have also been proposed using phage which selectively bind to the surface of a mammalian cell.
Further improvements have enabled the phage display system to express antibodies and antibody fragments on a bacteriophage surface, allowing for selection of specific properties, i.e., binding with specific ligands (EP 844306; U.S. Pat. Nos. 5,702,892; 5,658,727) and recombination of antibody polypeptide chains (WO 97/09436). A method to generate antibodies recognizing specific peptide-MHC complexes has also been developed (WO 97/02342). See also U.S. 5,723,287; 5,565,332; and 5,733,743.
U.S. Pat. No. 5,534,257 describes an expression system in which foreign epitopes up to about 30 residues are incorporated into a capsid protein of a MS-2 phage. This phage is able to express the chimeric protein in a suitable bacterial host to yield empty phage particles free of phage RNA and other nucleic acid contaminants. The empty phage are useful as vaccines.
The degree of expression of polypeptides as fusion proteins on the surface of bacteriophage particles is variable and depends, to some extent, on the size of the polypeptide. Conventional phage display systems use wild type phage coat proteins and fuse the heterologeous polypeptide to the amino terminus of the wild type amino acid sequence or an amino terminus resulting from truncation of the wild type coat protein sequence. Segments of linker amino acids have also been added to the amino terminus of the wild type coat protein sequence to improve selection and target binding.
Notwithstanding numerous modifications and improvements in phage technology, a need continues to exist for improved methods of displaying polypeptides as fusion proteins in phage display methods.
Methods of transforming cells to introduce new DNA are of great practical interest in molecular biology and modern genetic engineering. Early methods involved chemical treatment of bacteria with solutions of metal ions, generally calcium chloride, followed by heating to produce competent bacteria capable of functioning as recipient bacteria and able to take up heterologous DNA derived from a variety of sources. These early protocols provided transformation yields of about 105-106 transformed colonies per μgram of plasmid DNA. Subsequent improvements using different cations, longer treatment times and other chemical agents have allowed improvements in transformation efficiency of up to about 108 colonies/μgram of DNA. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., page 1.74.
Cells can also be transformed using high-voltage electroporation. Electroporation is suitable to introduce DNA into eukaryotic cells (e.g. animal cells, plant cells, etc.) as well as bacteria, e.g., E. coli. Sambrook et al., ibid, pages 1.75, 16.54-16.55. Different cell types require different conditions for optimal electroporation and preliminary experiments are generally conducted to find acceptable levels of expression or transformation. For mammalian cells, voltages of 250-750 V/cm result in 20-50% cell survival. An electric pulse length of 20-100 ms at a temperature ranging from room temperature to 0° C. and below using a DNA concentration of 1-40 μgram/mL are typical parameters. Transfection efficiency is reported to be higher using linear DNA and when the cells are suspended in buffered salt solutions than when suspended in nonionic solutions. Sambrook et al., above, pages 16.54-16.55.
Dower et al., 1988, Nucleic Acids Research, 16:6127-6145 extensively studied high-efficiency transformation of E. coli by high-voltage electroporation. This study evaluated numerous parameters, including electrical variables such as the effect of field strength and pulse length, the effect of DNA concentration and cell concentration on the recovery of transformants, accuracy, reproducibility, etc. and provided a protocol for high-efficiency electrotransformation of E. coli cells. The optimized protocol of Dower et al. uses cells concentrated in a range of at least 2 and up to 4×1010/mL, a DNA concentration of from about 1 to 10 μgrams/mL, 12.5-16.7 kV/cm, 3-25 μF and the electroporation is conducted at 0° C. (ice temperature). These studies were conducted with highly purified closed circular plasmid DNA, which is known to give high transformation efficiencies. Dower et al. report transformation efficiencies of 109-1010 transformants/μgram of DNA achieved by highly optimizing these parameters. For library formation, Dower et al. suggests using a DNA concentration of less than 10 nanogram/mL and a cell concentration of greater than 3×1010 to minimize co-transformants. See also U.S. Pat. Nos. 4,910,140 and 5,186,800 to Dower et al. and U.S. Pat. No. 4,849,355 to Wong.
Several attempts have been made to improve the design of electroporation apparatus (see, for example, U.S. Pat. Nos. 5,173,158; 5,098,843; 5,422,272; 5,232,856; and 5,283,194) and to improve electroporation of specific cells (see U.S. Pat. No. 5,128,257). U.S. Pat. No. 5,124,259 describes an improved buffer for electroporation. U.S. Pat. No. 4,956,288 describes a method for producing cells containing foreign DNA in high copy numbers.
The attainment of higher transformation efficiencies by optimizing the electroporation parameters has been difficult. The use of higher voltages and longer pulses results in an increase in cell death, decreasing the total number of transformed cells. Highly optimized electroporation still results in about 50-75% cell death. Dower et al. represents an important investigation of the parameters of electroporation and the protocol described in this paper has formed the basis of more recent electroporation procedures
An important emerging use of cell transformations, including electroporation, is the preparation of peptide and protein variant libraries. In these applications, a replicable transcription vector, for example a plasmid, is reacted with a restriction enzyme to open the plasmid DNA, desired coding DNA is ligated into the plasmid to form a library of vectors each encoding a different variant, and cells are transformed with the library of transformation vectors in order to prepare a library of polypeptide variants differing in amino acid sequence at one or more residues. The library of peptides can then be selectively panned for peptides which have or do not have particular properties. A common property is the ability of the variant peptides to bind to a cell surface receptor, an antibody, a ligand or other binding partner, which may be bound to a solid support. Variants may also be selected for their ability to catalyze specific reactions, to inhibit reactions, to inhibit enzymes, etc.
In one application, bacteriophage (phage), such as filamentous phage, are used to create phage display libraries by transforming host cells with phage vector DNA encoding a library of peptide variants. J. K. Scott and G. P. Smith, Science, (1990), 249:386-390. Phagemid vectors may also be used for phage display. Lowman and Wells, 1991, Methods: A Companion to Methods in Enzymology, 3:205-216. The preparation of phage and phagemid display libraries of peptides and proteins, e.g. antibodies, is now well known in the art. These methods generally require transforming cells with phage or phagemid vector DNA to propagate the libraries as phage particles having one or more copies of the variant peptides or proteins displayed on the surface of the phage particles. See, for example, Barbas et al., Proc. Natl. Acad. Sci., USA, (1991), 88:7978-7982; Marks et al., J. Mol. Biol., (1991), 222:581-597; Hoogenboom and Winter, J. Mol. Biol., (1992), 227:381-388; Barbas et al., Proc. Natl. Acad. Sci., USA, (1992), 89:4457-4461; Griffiths et al., EMBO Journal, (1994), 13:3245-3260; de Kruif et al., J. Mol. Biol., (1995), 248:97-105; Bonnycastle et al., J. Mol. Biol., (1996), 258:747-762; and Vaughan et al., Nature Biotechnology (1996), 14:309-314. The library DNA is prepared using restriction and ligation enzymes in one of several well known mutagenesis procedures, for example, cassette mutagenesis or oligonucleotide-mediated mutagenesis.
A recurring problem with transformation by electroporation, in particular with phage or phagemid vector DNA libraries, is the low transformation efficiency which has generally been in the range of 107-108 transformations/μgram of DNA. The low transformation efficiency has limited the size of libraries which can be prepared with a single electroporation step. Vaughan et al., above, describe a modified procedure in which several hundred electroporations were conducted to achieve a library with about 1010 recombinants.
Reaction mixtures obtained by enzymatic manipulation of DNA and RNA contain proteins, salts, etc., which are contaminants of the desired DNA or RNA. To obtain the purified nucleic acid, these mixtures are usually extracted with phenol/chloroform or similar solvent and then the DNA is precipitated with ethanol and resuspended in an appropriate amount of water or buffer to provide the DNA concentrations recommended by Dower et al. Bonnycastle et al., above, describe extracting a ligation reaction with chloroform/phenol/isoamyl alcohol followed by resuspension of the DNA in water and desalting by filtration over an exclusion membrane. This procedure allowed electroporation of electrocompetent MC1061 E. coli cells using a DNA concentration of about 20 μgrams/mL.
Despite two decades of research into electroporation and parameters affecting transformation efficiency, a need continues to exist for improved electroporation processes, in particular, for the transformation of cells with libraries of phage and phagemid DNA vectors.